Protein NirP1 regulates nitrite reductase and nitrite excretion in cyanobacteria

When the supply of inorganic carbon is limiting, photosynthetic cyanobacteria excrete nitrite, a toxic intermediate in the ammonia assimilation pathway from nitrate. It has been hypothesized that the excreted nitrite represents excess nitrogen that cannot be further assimilated due to the missing carbon, but the underlying molecular mechanisms are unclear. Here, we identified a protein that interacts with nitrite reductase, regulates nitrogen metabolism and promotes nitrite excretion. The protein, which we named NirP1, is encoded by an unannotated gene that is upregulated under low carbon conditions and controlled by transcription factor NtcA, a central regulator of nitrogen homeostasis. Ectopic overexpression of nirP1 in Synechocystis sp. PCC 6803 resulted in a chlorotic phenotype, delayed growth, severe changes in amino acid pools, and nitrite excretion. Coimmunoprecipitation experiments indicated that NirP1 interacts with nitrite reductase, a central enzyme in the assimilation of ammonia from nitrate/nitrite. Our results reveal that NirP1 is widely conserved in cyanobacteria and plays a crucial role in the coordination of C/N primary metabolism by targeting nitrite reductase.


Supplementary Figures
).The PCR of NirP1oex representative for all pVZ322-containing strains is shown.Abbreviations: M, marker; bp, base pairs; c., clone; -, negative control (water control); +, positive control (purified plasmid as template).This figure is an extension to Fig. 1 and illustrates technical aspects.Source data are provided as a Source Data file.These data represent an extension to the results shown in Fig. 2.

Fig. S1 .
Fig. S1.Generation of ΔnirP1 and nirP1-overexpressing strains.a The nirP1 locus in the wild type and in the ΔnirP1 deletion strain, as well as of a pVZ322 plasmid derivative harboring a nirP1 gene copy under the control of the Cu 2+ -inducible PpetE promoter in the nirP1 overexpression strains (NirP1oex and ΔnirP1::oex) and the complementation strain (ΔnirP1:: nirP1) under the control of its native promoter.The nirP1 gene was previously assigned to the transcriptional unit (TU) 2296 (1) extending from position 2215954 to 2217039 on the forward strand of the Synechocystis 6803 chromosome (GenBank accession no.NC_000911).The first segment of TU2296 has also been annotated as ncr1070 due to its sometimes-divergent accumulation compared to the second half, overlapping gene sll1864 on the reverse strand.In the ΔnirP1 strain, the gene was replaced by a streptomycin resistance cassette (Strep R ) via homologous recombination.The plasmid enabling ectopic nirP1 expression was introduced into Synechocystis wild type and ΔnirP1 strains.The red arrows indicate primer binding sites used to verify the mutants.b PCR verification of the genotypes of independently obtained mutant strains.Three clones were tested each, using primers P_AK9/P_AK10 (for ΔnirP1) or P_AK16/ P_AK17 (overexpression and complementation strains NirP1oex, ΔnirP1::oex and ΔnirP1::nirP1; primer sequences in TableS1).The PCR of NirP1oex representative for all pVZ322-containing strains is

Fig. S2 .
Fig. S2.Sequence similarities in the nirP1 promotor region and promotor binding motifs.a The promotor region (sequences from -300 to +10, with +1 as the TSS) of 456 nirP1 homologues from Supplementary Dataset 1 were used for sequence alignment.MEME was used to illustrate the conserved motifs.The NtcA binding motif is shown in a black box, the important nucleotides for transcription factor binding in a blue box.Details of the motifs are given in Supplementary Dataset 2. b Sequence alignment of the tandem repeat.c Sequence logo of 135 homologs that have the tandem repeat motif in Supplementary Dataset 2.These data represent an extension to the results shown in Fig.1and Fig.2.

Fig. S4 .Fig. S7 .
Fig. S4.Phenotypical differences between nirP1 mutants and the wild type.a Pigmentation phenotype of wild type and nirP1 overexpressor (NirP1oex) without and with 2 µM Cu2SO4 to trigger the expression of nirP1 for 24 h from the PpetE promoter.Lower panel: Repetition with three biological replicates of NirP1oex (R1 to R3).WT was used as negative control.b Drop dilution assay on solid medium comparing

. S3. NirP1 expression is mediated through NtcA and a Ci-sensitive promoter.
deprivation.After 7 days (time point 144 h) of nitrogen starvation, bleached cells were transferred back to standard 11 medium (indicated as Rec) to start the recovery.c Cells were grown in standard conditions T0 and were further grown with additional with 10 mM N 4Cl for 24 h.Significance was calculated with a two-sample t test with unequal variance (Welch's t test; , P < 0.05; , P < 0.01; , P < 0.001) between the strains at corresponding time points.Further details of statistical analysis are given in Supplementary Dataset 3.

Table S2 .
Plasmids used in this study.
pILA::PnirP1::luxAB Plasmid to fuse the nirP1 promotor region to luxAB genes for bioluminescence assay This study pILA::PNtcA-Mut::luxAB Plasmid to fuse the nirP1 promotor region with mutation in the NtcA binding site to luxAB for bioluminescence assay This study pILA::PRepeat-Mut::luxAB Plasmid to fuse the nirP1 promotor region with mutations in the upstream binding motif to luxAB for bioluminescence assay This study